Nodal is a transforming growth factor (TGF)-beta like protein that we discovered on the basis of a retroviral insertional mutation and showed to be essential for early development. Our studies on the nodalnull mutation revealed a major role in gastrulation. Our primary emphasis in the past few years has been on nodal signaling later in development and its role in left/right axial development. This work stems from our key observation that nodal is expressed asymmetrically around the midline node and only in the left lateral plate mesoderm of early somite stage vertebrate embryos. This asymmetric expression pattern has led us to investigate whether nodal regulates the development of organs such as the heart and lungs that are asymmetric in structure or position within the body. Because the nodal insertional mutation disrupts development at gastrulation prior to left/right development, we generated a nodal allele flanked by loxP sites (floxed) to allow us to conditionally delete nodal by crossing to transgenic mice expressing Cre recombinase at specific stages and/or tissues. Unexpectedly, but fortuitously, we discovered that the floxed nodal allele is hypomorphic. Embryos compound heterozygous for the floxed and a nodal null allele undergo gastrulation but then display abnormalities at later stages that fall into three distinct phenotypic classes. These results were found in the absence of any Cre mediated recombination. Comprehensive analysis of the phenotype of one mutant class has now provided conclusive evidence of the essential role nodal signaling plays in the proper left/right asymmetric development of the heart, lungs, vasculature and stomach. The reduced function of this hypomorphic nodal allele also has been extremely informative for furthering our understanding of nodal function in other developmental processes. Our study of the nodal hypomorph has established an essential function for nodal in the development of the anterior/posterior body axis and correct patterning of the forebrain. Our analysis has shown that nodal plays two roles in these processes. First, nodal signaling prior to gastrulation is essential for the movement of specific extraembryonic cells to a position adjacent to the future head region. These extraembryonic cells provide the initial neural inducing signal to embryonic cells that will become the forebrain. Nodal signaling then is required at a later stage to establish the node. The node is the source of cells that migrate to the head region, replacing the original extraembryonic cells, and provide a second neural inducing signal. Thus our analysis of the nodal hypomorph has provided key insight into two important developmental problems. Current knowledge of the ultimate outcome of nodal signaling in the process of mesoderm formation at the start of gastrulation is still limited. To further our understanding we have utilized P19 pluripotent embryonal carcinoma cells, which provide a cell culture model system for differentiation events occurring during early development. We found that P19 cells overexpressing nodal differentiate into mesoderm. We exploited the responsiveness of P19 cells and the availability of recombinant nodal protein to dissect the intracellular components of the nodal signaling pathway. Our studies show that nodal signals through Smad2 and Smad3. These Smads are also used by TGF-beta and activin, another TGF-beta like factor. However, we also have shown that nodal signaling differs from TGF-beta and activin signaling in requiring the function of an extracellular protein of the EGF-CFC family, either Cripto or Cryptic. Having developed the capacity to generate recombinant nodal protein in our laboratory we are now addressing the physical interaction of nodal with candidate receptors and with EGF-CFC proteins. We are also exploiting the P19 system to determine how the gene expression profiles of early embryonic cells change in response to nodal. We have compared the P19 cells that overexpress nodal with normal P19 cells in a screen of micro-arrays of 20,000 mouse genes expressed in the developing embryo. This study has revealed a number of genes whose expression differs significantly in these two cell populations. We are determining whether any of these are direct nodal target genes by analyzing expression in P19 cells briefly treated with protein and expression in the developing wild-type and nodal mutant embryo. For instance, we identified HoxB1 as a gene downregulated in P19 cells overexpressing nodal. Early HoxB1 expression in the normal embryo completely overlaps that of nodal and is abnormally low in the nodal hypomorphic mutant. This is one example of how the microarray approach will help us to develop novel insights into the biological consequences of nodal signaling.